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Structural features stabilized by divalent cation coordination within hepatitis E virus ORF1 are critical for viral replication.

ABSTRACT: Hepatitis E virus (HEV) is an RNA virus responsible for over 20 million infections annually. HEV's open reading frame (ORF)1 polyprotein is essential for genome replication, though it is unknown how the different subdomains function within a structural context. Our data show that ORF1 operates as a multifunctional protein, which is not subject to proteolytic processing. Supporting this model, scanning mutagenesis performed on the putative papain-like cysteine protease (pPCP) domain revealed six cysteines essential for viral replication. Our data are consistent with their role in divalent metal ion coordination, which governs local and interdomain interactions that are critical for the overall structure of ORF1; furthermore, the 'pPCP' domain can only rescue viral genome replication in trans when expressed in the context of the full-length ORF1 protein but not as an individual subdomain. Taken together, our work provides a comprehensive model of the structure and function of HEV ORF1.

SUBMITTER: LeDesma R

PROVIDER: S-EPMC9977285 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Structural features stabilized by divalent cation coordination within hepatitis E virus ORF1 are critical for viral replication.

LeDesma Robert R Heller Brigitte B Biswas Abhishek A Maya Stephanie S Gili Stefania S Higgins John J Ploss Alexander A

eLife 20230228

Hepatitis E virus (HEV) is an RNA virus responsible for over 20 million infections annually. HEV's open reading frame (ORF)1 polyprotein is essential for genome replication, though it is unknown how the different subdomains function within a structural context. Our data show that ORF1 operates as a multifunctional protein, which is not subject to proteolytic processing. Supporting this model, scanning mutagenesis performed on the putative papain-like cysteine protease (pPCP) domain revealed six ...[more]

PMID: 36852909

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Project description:Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis and jaundice in the world. Current understanding of the molecular virology and pathogenesis of hepatitis E is incomplete, due particularly to the limited availability of functional tools. Here, we report the development of tagged HEV genomes as a novel tool to investigate the viral life cycle. A selectable subgenomic HEV replicon was subjected to random 15-nucleotide sequence insertion using transposon-based technology. Viable insertions in the open reading frame 1 (ORF1) protein were selected in a hepatoblastoma cell line. Functional insertion sites were identified downstream of the methyltransferase domain, in the hypervariable region (HVR), and between the helicase and RNA-dependent RNA polymerase domains. HEV genomes harboring a hemagglutinin (HA) epitope tag or a small luciferase (NanoLuc) in the HVR were found to be fully functional and to allow the production of infectious virus. NanoLuc allowed quantitative monitoring of HEV infection and replication by luciferase assay. The use of HA-tagged replicons and full-length genomes allowed localization of putative sites of HEV RNA replication by the simultaneous detection of viral RNA by fluorescence in situ hybridization and of ORF1 protein by immunofluorescence. Candidate HEV replication complexes were found in cytoplasmic dot-like structures which partially overlapped ORF2 and ORF3 proteins as well as exosomal markers. Hence, tagged HEV genomes yield new insights into the viral life cycle and should allow further investigation of the structure and composition of the viral replication complex.IMPORTANCE Hepatitis E virus (HEV) infection is an important cause of acute hepatitis and may lead to chronic infection in immunocompromised patients. Knowledge of the viral life cycle is incomplete due to the limited availability of functional tools. In particular, low levels of expression of the ORF1 protein or limited sensitivity of currently available antibodies or both limit our understanding of the viral replicase. Here, we report the successful establishment of subgenomic HEV replicons and full-length genomes harboring an epitope tag or a functional reporter in the ORF1 protein. These novel tools should allow further characterization of the HEV replication complex and to improve our understanding of the viral life cycle.

Project description:Hepatitis E virus (HEV) is a clinically important positive-sense RNA virus. The ORF1 of HEV encodes a nonstructural polyprotein of 1,693 amino acids. It is not clear whether the ORF1 polyprotein (pORF1) is processed into distinct enzymatic domains. Many researchers have attempted to understand the mechanisms of pORF1 processing. However, these studies gave various results and could never convincingly establish the mechanism of pORF1 processing. In this study, we demonstrated the possible role of thrombin and factor Xa in pORF1 processing. We observed that the HEV pORF1 polyprotein bears conserved cleavage sites of thrombin and factor Xa. Using a reverse genetics approach, we demonstrated that an HEV replicon having mutations in the cleavage sites of either thrombin or factor Xa could not replicate efficiently in cell culture. Further, we demonstrated in vitro processing when we incubated recombinant pORF1 fragments with thrombin, and we observed the processing of pORF1 polyprotein. The treatment of a liver cell line with a serine protease inhibitor as well as small interfering RNA (siRNA) knockdown of thrombin and factor Xa resulted in significant reduction in the replication of HEV. Thrombin and factor Xa have been well studied for their roles in blood clotting. Both of these proteins are believed to be present in the active form in the blood plasma. Interestingly, in this report, we demonstrated the presence of biologically active thrombin and factor Xa in a liver cell line. The results suggest that factor Xa and thrombin are essential for the replication of HEV and may be involved in pORF1 polyprotein processing of HEV.IMPORTANCE Hepatitis E virus (HEV) causes a liver disorder called hepatitis in humans, which is mostly an acute and self-limiting infection in adults. A high mortality rate of about 30% is observed in HEV-infected pregnant women in developing countries. There is no convincing opinion about HEV ORF1 polyprotein processing owing to the variability of study results obtained so far. HEV pORF1 has cleavage sites for two host cellular serine proteases, thrombin and factor Xa, that are conserved among HEV genotypes. For the first time, this study demonstrated that thrombin and factor Xa cleavage sites on HEV pORF1 are obligatory for HEV replication. Intracellular biochemical activities of the said serine proteases are also essential for efficient HEV replication in cell culture and must be involved in pORF1 processing. This study sheds light on the presence and roles of clotting factors with respect to virus replication in the cells.

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Structural features stabilized by divalent cation coordination within hepatitis E virus ORF1 are critical for viral replication.

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Structural features stabilized by divalent cation coordination within hepatitis E virus ORF1 are critical for viral replication.

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